Journal of International Dental and Medical Research ISSN 1309-100X MTA1 Expression in Salivary Mucoepidermoid

http://www.jidmr.com Omar I. Ahmed and et al

MTA1 Expression in Salivary Mucoepidermoid Carcinoma: with Special Emphasis

on Grading Systems
Omar I. Ahmed1*, Lehadh M. AL-Azzawi2
1. Department of dentistry, Bilad Al- Rafidain university, Diyala,Iraq.
2. Department of dentistry, Al- Bayan University, Baghdad, Iraq.

Abstract
Metastasis-associated protein-1 (MTA1) has been recently identified as a unique gene which
plays a key role in tumorigenesis and progression of cancer cells. The object behind this study is to
evaluate MTA1 immunoexpression and its relationship with predictive value of point- based grading
systems in salivary mucoepidermoid carcinoma. A total of 22 formalin-fixed, paraffin-embedded
specimens of mucoepidermoid carcinoma were prepared for immunohistochemical staining with
MTA1 antibodies. Assessment of MTA1 immunostaining was achieved by counting the proportion
of positively-stained tumor cells in 5 high power fields; and staining was analyzed in relation to
tumor grading systems and other clinicopathologic features. MTA1 showed nuclear and cytoplasmic
expression in varying intensity in 95% of cases. Non- significant correlation was found between
MTA1expression and age, gender, site of the tumor (p > 0.05). However, statistically significant
correlation was observed between MTA1expression and clinical stage, as well, to nodal
involvement (p = 0.009 and 0.007; respectively). Regarding histologic grade, high MTA1 level was
significantly associated with grade of tumors categorized by Auclair and Brandwein systems (p =
0.001 and 0.009; respectively). MTA1 expression significantly correlates with tumor grade and
progression, and it has a potential role to predict the prognosis of salivary MEC.
Experimental article (J Int Dent Med Res 2019; 12(4): 1253-1257)
Keywords: Salivary mucoepidermoid carcinoma, MTA1, Tumor grade, Metastasis.
Received date: 20 August 2018 Accept date: 18 January 2019

Introduction the high MTA1 expression is significantly

correlated with tumor invasion and progression.7,8
Mucoepidermoid carcinoma is the most However, high immunoexpression has been
common salivary gland malignancy; ascertained for MTA1protein in different tumor
approximately accounting for 30% of these types, and it has statistically significant
tumors.1 MEC exhibits a complex histopathologic correlation with tumor grade and
features; and it is generally categorized into low, angiogenesis.9,10 The purpose of this study is to
intermediate, or high grade.2,3 evaluate the correlation between MTA1
Metastasis-associated protein-1 (MTA1), immunoexpression and grade of salivary MEC
a unique member of the MTA proteins, which is categorized by Auclair and Brandwein systems.
specifically involved with more aggressive cell
phenotype and tumor metastastasis.4 MTA1- Materials and Methods
protein is transcriptional co-repressors involved
with complex-containing deacetylation molecules This study included 22 formalin-fixed,
that may inactivate the p53 protein.4,5 Moreover, paraffin-embedded specimens of salivary MEC
MTA1 may induct aberrant tumor that retrieved from the archives of the
neovascularization via stabilizing hypoxia- Department of Oral Diagnosis/College of
inducible factor-1α.6 It has been suggested that Dentistry/ Baghdad University. Clinical data
concerning the age, gender, site of tumor and
lymph node metastasis was obtained from
*Corresponding author: patients’ medical records for the period extending
Omar I Ahmed from 2009 to 2017. For all specimens, 4µm thick
University of Baghdad, college of dentistry,
Baghdad, Iraq.
sections were prepared and stained with
E-mail: Info@baghentistry.com hematoxylin and eosin (H&E) stain to confirm the
diagnosis. Clinicopathologic characteristics of the

Volume ∙ 12 ∙ Number ∙ 4 ∙ 2019 Page 1253

 Journal of International Dental and Medical Research ISSN 1309-100X MTA1 Expression in Salivary Mucoepidermoid
http://www.jidmr.com Omar I. Ahmed and et al

overall series are summarized in (Table 1). The Negative controls were achieved by omitting the
histological grade of tumors was evaluated primary antibody. After washing with phosphate
according to criteria of Auclair et al. (1992), and buffer saline (PBS), the tissue sections were
that proposed by Brandwein et al, (2001), which incubated with biotin-free, anti-rabbit secondary
are approved by The WHO tumor antibody conjugated with horseradish peroxidase
classification.11,12 Moreover, all cases were (HRP) for 15 min, and then color was developed
staged according to the 7th edition of the using 3,3’-diaminobenzidine as chromogen. then,
American Joint Committee on Cancer (AJCC).13 it is counterstained with Mayer’s hematoxylin,
mounted and covered with cover slip for
Total evaluation using microscope (OLYMPUS, Japan).
Variables
No. (%)
Age group (years) IHC scoring and Statistical analysis
The degree of IHC staining was
<40 5 22.7
separately evaluated by two pathologists who
>40 17 77.3 were blinded to the clinicopathologic information.
Gender MTA1 immunostaining was scored by calculating
Female 12 54.5 the proportion of positively stained tumor cells in
Male 10 45.4 5 microscopic high power fields that reveal higher
Site immunopositivity as follows: Score 0 (0-5%
Major 11 50 positive cells); Score I (6-25% positive cells);
Minor 11 50 score II (26-50% positive cells); score III (51-75%
Clinical stage
positive cells) and Score IV (≥76 positive cells). A
Mann-Whitney test, Kruskal Wallis test and
I 9 40.9
Spearman´s correlation coefficient test were
II 7 31.8 used to compare the result between groups and
III 3 13.6 the relation with clinical-pathological parameters
IV 3 13.6 such as patient age, gender, tumor site,
Lymph node status metastasis to lymph nodes, tumor grade and
-ve 16 72.7 clinical stage. We used the SPSS version 24
+ve 6 27.3 software to statistically analyze the data. P-
Auclair grading system values <0.05 were considered statistically
15 68.1 significant in all cases.
Low grade
High grade 7 31.9
Brandwein grading ststem Results
6 27.3
I
6 27.3 Representative images of MTA1
II
10 45.4
III immunohistochemical (IHC) expression in MEC
Table 1. Clinical-pathological Parameters of the are shown in (Figure 1A-F). Positive
Overall Series. immunostaining of MTA1 was observed in both
nuclear and cytoplasmic compartments in (95%)
Immunohistochemistry (IHC) and of the MEC tissues. While, negative or weak
analysis MTA1stanining was found in the adjacent non-
The sections in these series were cancerous ductal epithelial tissues in the same
deparaffinized in xylene, rehydrated through section (Figure 1 A).
graded alcohols, immersed in 0.3% H2O2 for 20
min to inhibit endogenous peroxidase activity,
and antigen retrieval performed using citrate
buffer with PH = 6. Nonspecific binding was
blocked with 1% serum albumin at room
temperature for 10 min, then the sections were
incubated with anti-MTA1 rabbit-polyclonal
antibody (1:500 dilution; Abcam, Cambridge, UK)
overnight at 4 °C in a humidified chamber.

Volume ∙ 12 ∙ Number ∙ 4 ∙ 2019 Page 1254

 Journal of International Dental and Medical Research ISSN 1309-100X MTA1 Expression in Salivary Mucoepidermoid
http://www.jidmr.com Omar I. Ahmed and et al

Brandwein grading systems, the median MTA1

score was significantly higher among tumors with
high grade (score-3) compared to those with low
grade (median score = 2), thus a statistically
significant correlation was observed between
MTA1 expression and grade of tumors
categorized by Auclair and Brandwein systems (p
value < 0.001 and 0.009; respectively), as in
(Figure 2).

MTA1 scores* Median

Parameters MTA p-value†
0 I II III IV score

Age group
(years)
0 1 3 1 0 2 0.15
<40
1 3 6 5 2 3 [NS]
>40
Gender
Male 0 1 4 3 2 3 0.10
Female 1 3 5 3 0 2 [NS]

Site
Minor 0 1 6 3 1 2 0.45
Figure 1. Immunohistochemical analysis of Major 1 3 3 3 1 2 [NS]
MTA1 protein in salivary MEC; (A) Negative
Clinical Stage
staining in normal salivary ductal tissue (40x); (B) I-II 1 4 8 3 0 2
low grade tumor shows cystic components lined 0.009
III-IV 0 0 1 3 2 4
by mucous and intermediate cell: brown stain
Lymph node
(40x); (C) MEC, with small islands of metastasis
intermediate and epidermoid cell, mainly N0 1 4 8 3 0 2
cytoplasmic expression (20x); (D) High grade N1 0 0 0 1 2 4 0.007
N2 0 0 1 2 0 3
MEC shows nuclear MTA1expression (40x); (E)
Solid tumor with sheets of anaplastic epidermoid Auclair
cells with cytoplasmic expression (40x); (F) grading
system
lymph node infiltrated with tumor cells expressing Low 1 4 9 1 0 2 0.001
nuclear MTA1 protein (20x). High 0 0 0 5 2 3
Brandwein
Correlation between MTA1 expression and grading
system
the clinicopathologic features of salivary MEC Low 1 2 3 0 0 2 0.009
Table 2 showed the correlation between Intermediate 0 2 3 1 0 2
MTA1 expression and the clinicopathologic High 0 0 3 5 2 3
* Score 0 (0-5% positive cells); Score I (6 - 25% positive cells);
characteristics in all series. Accordingly, no score II (26 - 50% positive cells); score III (51 - 75% positive cells)
statistically significant correlation between MTA1 and Score IV (≥ 76 positive cells). [NS] Non- significant p > 0.05; †
protein expression and clinicopahtologic features, A Mann-Whitney test, and Kruskal-Wallis test.
such as age, gender and tumor site (P > 0.05). Table 2. Correlation Between MTA1 Expression
However, the expression level of MTA1 protein and the Clinicopathologic Features.
was found to be significantly associated with
clinical stage, showing a lower expression
pattern in early-stage disease (I and II), and a
stronger expression (median MTA1 score 4), in
late stages (III and IV; P = 0.009). High MTA1
expression also was found to be significantly
associated with positive nodal metastasis
(p=0.007). Regarding both Auclair and

Volume ∙ 12 ∙ Number ∙ 4 ∙ 2019 Page 1255

 Journal of International Dental and Medical Research ISSN 1309-100X MTA1 Expression in Salivary Mucoepidermoid
http://www.jidmr.com Omar I. Ahmed and et al

consuming and each of systems vary regarding

to the behavior of each particular grade. Although,
a various methods were utilized as avenue in
salivary cancer monitoring, it is necessary to find
a new biomarkers that specifically predict tumor
aggressiveness and behavior.14,15,16 In our study,
MTA1 exhibited different IHC and subcellular
distribution which was in consistence with other
researches.17,18 In the present study, we
evaluated MTA1 expression in salivary MEC, and
there was no statistically significant correlation
between MTA1 level and clinical findings, such
as patient age, gender and tumor site. This
finding was in agreement with previous
studies.19,20 In this study, also we observed that
MTA1expression was significantly correlated with
clinical stage and lymph node metastasis. In this
context, our findings agree with other researches,
in which IHC analysis indicated that MTA1
expression was significantly correlated with
tumor aggressiveness, and positive nodal status
in head and neck cancer.21,22 However, our
results were not in conformity with findings from
Andishehtadbir et al. (2016) who found that
MTA1 protein expression had no significant
statistical correlation with clinical stage, lymph
node status and metastasis of salivary MEC
which may be attributable to an insufficient
number of cases.23 On the other hand, in this
study, a significant cohort of cases showed a
nuclear MTA1 expression compared to
cytoplasmic one, and this suggest the invasive
growth and metastatic potential of these
tumors.24 However, our explanation for
cytoplasmic expression of MTA1 based on earlier
investigations which have been stated that
MTA1s, a short version of cytoplasmic MTA1
may bind to estrogen receptor-alpha (ER-α) and
inhibit its nuclear function by non-genomic
activity of (ER-α) that occurs in cytoplasm of
cancer cells. Thus, this may rationalize the
aggressive behavior of these tumors. 25,26
Regarding tumor grading, we found a significant
Figure 2. Diagram Shows MTA1 Expression up-regulation of MTA1 expression in higher
(Median Scores) Regarding (A) Auclair; (B) grade tumors classified according to both Auclair
Brandwein System. and Brandwein system with strong positive linear
correlation between MTA1 score (r = 0.7) and
Discussion Brandwein tumor grade. This result indicated a
strong association of MTA1 expression with the
In the broad sense, all grading systems of progression of MEC. In contrast to our findings,
MEC are prognostically effective and show a Andishehtadbir et al. (2016), found no correlation
quite reproducibility among pathologists, between MTA1 expression and histologic grade
although they are somewhat complex, time- of salivary MEC, although high MTA1 expression

Volume ∙ 12 ∙ Number ∙ 4 ∙ 2019 Page 1256

 Journal of International Dental and Medical Research ISSN 1309-100X
http://www.jidmr.com
was seen in some cases with advanced tumor
size and clinical stage.23 The possible
explanation behind this disagreement is that, in
our analysis we have approved a standardized
grading systems for tumor classification, with
focus on more objective criteria, as compared to
previous study which was built on a subjective
morphologic and histologic features.23 In
conclusion, MTA1 expression significantly
correlates with histologic grade and progression
of salivary MEC, which means it may be
prognostically effective and has a significant
predictive value in salivary MEC.
Acknowledgements
The Authors wish to thank cancer
research institution, and staff of oral diagnosis
department, Baghdad College of dentistry for
supporting as a technical assistance. All authors
have made substantive contribution to this study,
and all have reviewed the final paper prior to its
submission. Regarding conflicting interest, the
authors have no funding or financial support to
declare.
Conflict of Interest
The authors declare that there is no
conflict of interest regarding the publication of
this article.
References
1. Luna MA. Salivary mucoepidermoid carcinoma: revisited. Adv
Anat Pathol 2006;13(6):293-307.
2. Eveson JW and Nagoa T. Disease of salivary glands. In:
Barnes L Surgical Pathology of Head and Neck. 3rd ed.
Pennsylvania: Informa health care, USA 2009;(1):547-50.
3. Seethala RR. Histologic Grading and Prognostic Biomarkers
in Salivary Gland Carcinomas. Adv Anat Pathol
2011;18(1):29-45.
4. Toh Y, Pencil SD, Nicolson GL. A novel candidate
metastasis-associated gene, mta1, differentially expressed in
highly metastatic mammary adenocarcinoma cell lines. cDNA
cloning, expression, and protein analyses. J Biol Chem
1994;269 (37):22958–63.
5. Toh Y, Nicolson GL. The role of the MTA family and their
encoded proteins in human cancers: molecular functions and
clinical implications. Clin Exp Metastasis 2009;26(3):215-27.
6. Yoo YG, Kong G, Lee MO. Metastasis-associated protein 1
enhances stability of hypoxia inducible factor-1alpha protein
by recruiting histone deacetylase 1. EMBO
2006;25(6):1231-41.
7. Sen N, Gui B, Kumar R. Role of MTA1 in Cancer Progression
and Metastasis. Cancer Metastasis Rev. 2014;33(4):879–89.
8. Yu C, Chen H, Wang J, et al. Nuclear expression of
metastasis-associated protein 1 (MTA1) is inversely related to
progression of oral squamous cell carcinoma in Taiwan. J
Dent Sci 2007;2(1):1-10.
Volume ∙ 12 ∙ Number ∙ 4 ∙ 2019
MTA1 Expression in Salivary Mucoepidermoid
Omar I. Ahmed and et al
9. Dias S, Zhou X, Ivanovic M, et al. Nuclear MTA1
overexpression is associated with aggressive prostate cancer,
recurrence and metastasis in African Americans. Sci Rep
2013;3:2331.
10. Jang k, Paik S, Chung H, Oh Y, Kong G. MTA1
overexpression correlates significantly with tumor grade and
angiogenesis in human breast cancers. Cancer Sci
2006;97(5):375.
11. Auclair PL, Goode RK, Ellis GL. Mucoepidermoid carcinoma
of intraoral salivary glands: evaluation and application of
grading criteria in 143 cases. Cancer 1992;69(8):2021-30.
12. Brandwein MS, Ivanov K, Wallace DI, et al. Mucoepidermoid
carcinoma. A clinicopathological study of 80 patients with
special reference to histological grading. Am J Pathol
2001;25(7):835-45.
13. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti
A, editors. AJCC cancer staging manual (7th ed). New York,
NY: Springer 2010;17:1471-74.
14. Seethala RR. An update on grading of salivary gland
carcinomas. Head Neck Patho 2009;3(1):69-77.
15. Seethala RR, Bennett A, Arrosi AA, Dvison JM, Krasinkas AM,
Hunt JL. Reproducibility of grading in salivary gland
mucoepidermoid carcinoma and correlation with outcome:
does system really matter? Mod Pathol 2008;21(1):241-1A.
16. Laidi F, Zaoui F. saliva diagnostic and cancer monitoring:
overview. J Int Dent Med Res 2015;5(2):94-7.
17. Liu J, Haijuan W, Changzhi H, Haili Q. Subcellular localization
of MTA proteins in normal and cancer cells. Cancer
Metastasis Rev 2014a;33(4):843-56.
18. Liu J, Haijuan W, Changzhi H, Haili Q. The subcellular
distribution and function of MTA1 in cancer differentiation.
Oncotarget 2014b;5(13):5153-64.
19. Li W F, Liu N, Xue RX, et al. Nuclear overexpression of
metastasis-associated protein 1 correlates significantly with
poor survival in nasopharyngeal carcinoma. J Trans Med
2012;10(78):1-9.
20. Liu T, Yang M, Yang S, Ge T, Gu L, Lou G. Metastasis-
associated protein 1 is a novel marker predicting survival and
lymph nodes metastasis in cervical cancer. Hum Pathol
2013;44(10):2275–81.
21. Kawasaki G, Yanamoto S, Yoshitomi I, Yamada S, Mizuno A:
Overexpression of metastasis-associated MTA1 in oral
squamous cell carcinomas: correlation with metastasis and
invasion. Int J Oral Maxillofac Surg 2008;37(11):1039-46.
22. Yuan T, Zhang H, Liu B, et al. Expression of MTA1 in
nasopharyngeal carcinoma and its correlation with prognosis.
Med Oncol 2014;31(12):330.
23. Andisheh-Tadbir A, Dehghani-Nazhvani A, Ashraf MJ,
Khademi B, Mirhadi H, Torabi-Ardekani S. MTA1 Expression
in Benign and Malignant Salivary gland Tumors. Iran J Otorhi
2016;28(1):51-9.
24. Andisheh-Tadbir A, Dehghani-Nazhvani A, Pardis S, et al.
Metastasis-Associated Protein 1 Expression in Oral
Squamous Cell Carcinomas: Correlation with Metastasis and
Angiogenesis. Turk Pathol Derg 2015;31(2):9-15.
25. Kumar R, Wang RA, Mazumdar A, et al. A naturally occurring
MTA1 variant sequesters oestrogen receptor-alpha in the
cytoplasm. Nature 2002;418(6898):654.
26. Balasenthil S, Broaddus RR, Kumar R. Expression of
metastasis-associated protein 1 (MTA1) in benign
endometrium and endometrial adenocarcinomas. Human
Pathol 2006;37(6):656-61.
J
Page 1257